Stimulated Emission Depletion (STED) microscopy is one of the most important recent developments in light microscopy (Willig et al., 2006, Nature 440:935-9). STED allows for imaging cellular elements with diffraction-unlimited resolution; in practical terms, the resolution (normally limited to ~200-300 nm) is improved down to 30-60 nm. Together with the development of two-color STED microscopy (Donnert et al., 2007, Biophys J. 92:L67-9), this technique allows experimenters to pinpoint the position of various cellular elements with nanometer precision. Obtaining a cellular nanomap is not feasible with conventional light microscopy, due to its low resolution. Electron microscopy cannot be applied, as its labeling efficiency it too low. I propose here to use STED microscopy to characterize the positions of the major components of the synapse. The preparation will be cultured hippocampal neurons, which have numerous small (about one micron in diameter) synaptic nerve terminals. I will determine the locations of synaptic proteins involved in neurotransmitter release, in membrane retrieval and in pre- and post-synaptic active zone structure. Less specialized elements such as the cytoskeleton, mitochondria and endosomes of the synapse will also be investigated. The work will provide answers for a number of questions in the neuroscience field, such as how and where the synaptic vesicles get retrieved, how pre- and post-synaptic active zone elements correlate, and what the role of cytoskeletal elements is in synaptic transmission. The small size and relatively low complexity (compared to whole cells) of the synaptic boutons will allows the work to be completed within a reasonable timeframe. Successful completion of the project will encourage researchers to perform larger scale cellular nano-maps, which would eventually replace the largely erroneous cellular fractionation techniques currently used nowadays to determine the location of various proteins.